Acylation reactions with cyclic imides

Acylation reactions with cyclic imides

173 BtOCHIMICA ET BIOPHYSICA ACTA BBA 3 5 2 7 I ACYLATION REACTIONS WITH CYCLIC IMIDES DEREK G. SMYTH AND HANS TUPPY The National Institute for Medi...

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173

BtOCHIMICA ET BIOPHYSICA ACTA BBA 3 5 2 7 I

ACYLATION REACTIONS WITH CYCLIC IMIDES DEREK G. SMYTH AND HANS TUPPY The National Institute for Medical Research, Mill Hill, London, N.W.7 (Great Britain) and from the Institut fiir Biochemie, Wasagasse, Vienna I X (Austria)

(Received June ioth, 1968)

SUMMARY

Maleimide reagents have been examined for a potential use in the cross-linking of amino and thiol groups in proteins. The adducts obtained by reaction of N-ethylmaleimide or of N-(4-dimethyl-3,5-dinitroaminophenyl)maleimide with cysteine, homocysteine, and glutathione were prepared and the rates of reaction of the imide rings with water and with amino groups were studied. In the cysteine-maleimide addition products, where amino and thiol groups are located in positions sterically favourable for cross-linking, intramolecular aminolysis occurs readily. In contrast, the amino group of the homocysteine and glutathione adducts is comparatively stable.

Acylation reactions with cyclic imides

Previous studies on the reactions of N-ethylmaleimide with cysteine x, and of N-(4-dimetbyl-3,5-dinitroaminophenyl)maleimide (DDPM) with cysteine or with proteins containing thiol and amino groups ~, have shown that the reaction products can undergo intramolecular transamidation to form thiazine derivatives. A similar intramolecular acylation has been postulated in experiments on the reaction of

CO2H j c I. \ NI-I2

CH2SH

R'N

CO

CO

//" CM

io2H jcH\ NH 2

R.N-CO CO CH 2

CH2

.S

~o2H jell\ Nil

CO~ CO-CH 2 INHR

CH 2

/.S H

N-ethylmaleimide with hemoglobin 3. The present study is concerned with investigating the steric conditions that are compatible with the use of maleimide reagents for the intramolecular cross-linking of thiol and amino groups in model compounds. If the relative positioning of the two groups should prove to be critical for cross-linking, the reagents could prove valuable in investigations of protein conformation; certain Abbreviation: DDPM, N-(4-dimethyl-3,5-dinitroaminophenyl)maleimide. Biochim. Biophys. Acta, i68 (1968) I73-I8O

174

]). G. SMYTH, H. TUPPY

cysteine and lysine residues far apart in the linear sequence of protein could be identified as being closely positioned in space. To examine this possibility, we have isolated the products obtained by addition of N-ethyhnaleimide or of DDPM to the thiol group of cysteine, homocysteine, and glutathione and we have investigated the degree to which the derivatives undergo intramolecular aminolysis. Such transanfidation reactions would lead to the formation of 6, 7 and IX membered rings, respectively. In addition, the potential capacity of the maleimide-thiol adducts to act as intermolecular acylating agents has been examined by studying the reactivity of the imide ring of the addition product of N-ethylmaleimide with thioglycolic acid, and of DDPM with N-acetyl-L-cysteine, towards the anfino group of triglycine. RESULTS AND DISCUSSION

When a maleimide adduct of cysteine, homocysteine, or glutathione is maintained in neutral or alkaline solution, three competing reactions involving the imide ring may take place : hydrolysis, intramolecular aminolysis, and intermolecular aminolysis. Hydrolysis of the imide ring results in a release of H+ by ionisation of the formed carboxyl group ; provided no change occurs in the pK of the associated amino group, one H + is released corresponding to each imide ring undergoing hydrolysis. Aminolytic

~O2N

/CH NH2

I O2H jcH\ NH2

+

H+

C

R NN OC'CH2''~" ~ H

~N2

R.N-CO

iH2

ooc~ ~ s

S

J~ CO-CH 2

H

CO2H

~

jCH~ NH2

i H2 +

R NN-CO

~

N+

S

jc -OOC-CH 2

~4

reactions, both intra- and intermolecular, take place between imide carbonyl groups and nucleophilic amino groups. This reaction leads to a displacement of the equilibrium between charged and uncharged amino groups and may be accompanied by a release of H+; the extent to which H+ is liberated depends on the pH of the solution and the pK of the - N H + group. R--NH +

~

R'--NN2

+

I Maleimide

R/" CONHR Biochim. Bioph3's. Acla, 168 (I968) t73-lSO

H+

ACYLATION WITH CYCLIC IMIDES

175

TABLEI DECOMPOSITION OF MALEIMIDE--THIOL ADDITION PRODUCTS

3 mM solutions were titrated at constant pH with o.i M NaOH. Experiments were observed at 30.5 ° for at least 3 times the relevant half life period. Each recorded result is the mean of experiments performed in triplicate in which the greatest observed deviation was 5 %.

Substance

pH

K x IO a t t (rain -1) (rain)

N-Ethylmaleimide Cysteine-N-ethylmaleimide adduct Cysteine-N-ethylmaleimide adduct Homocysteine-N-ethylmaleimide adduct Glutathione-N-ethylmaleimide adduct N-Ethylmaleimide~hioglycolic acid adduct N-Ethylmaleimide-thioglycolic acid adduct N-Ethylmaleimide-thioglycolic acid adduct + triglycine (i : I) DDPM Cysteine-DDPM adduct Homocysteine-DDPM adduct Glutathione-DDPM adduct DDPM-N-acetylocysteine adduct DDPM-N-acetylocysteine adduct + triglycine (I :i)

8.5 8.5 9.5 9.5 9.5 8. 5 9.5 9.5 7.0 7 .o 7.0 7.0 7.0 7.0

27 5.6 26 4.1 4 .0 0.22 2-3 2.8 18. 7 43. i 2.74 2.8 1.o4 1.2

25.3 125 27.o 168 176 323 ° 3ol 248 37.2 16.2 253 249 671 578

The reactions undergone by the eight maleimide adducts were studied with aid of a p H stat ( R a d i o m e t e r Inc. Model T T T I a ) , which allowed m e a s u r e m e n t of r a t e s o f f o r m a t i o n o f H +. D i l u t e N a O H w a s a d d e d a u t o m a t i c a l l y t o m a i n t a i n t h e at the required value and the rates of addition were recorded graphically. From

the the pH the

TABLE II CHROMATOGRAPHY

OF

MALEIMIDE--THIOL

ADDITION

PRODUCTS

AFTER

INCUBATION

AT A L K A L I N E

pH VALUES Reaction mixtures were analysed by paper chromatography, as described in EXPERIMENTAL.

Reactants

RF of reactant

RF values of ninhydrinpositive products

Thioglycolic acid-N-ethylmaleimide adduct (pH 9.5) 0.84 Thioglycolic acid-N-ethylmaleimide adduct + triglycine (I:I) (pH 9.5) 0.84 0.I0

Cysteine-N-ethylmaleimide adduct (pH 8.5) Cysteine-N-ethylmaleimide adduct (pH 9.5) Homocysteine-N-ethylmaleimide adduct (pH 9.5) Glutathione-N-ethylmaleimide adduct (pH 9.5)

RF values of sulfurcontaining product

0.74 o.74 O.IO

0.27

0.21

o.58

0.27

o.16

0.58 o.16

o.41

0.30 o.16

0.70 0.30

o.I6 o.27

O.17

o.18

o.16

Biochim. Biophys. Acta, 168 (1968) 173-18o

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l). G. S M Y T H , H. T U P P V

curves obtained it was found that first-order kinetics were obeyed in all cases, the slope of the first-order plots permitting calculation of the reaction constants given in Table I. In addition, the composition of the reaction mixture obtained on incubation of each N-ethylmaleimide adduct was examined by paper chromatography, and these results are indicated in Table II. The addition product of cysteine with N-ethyhnaleimide at pH 8.5, 3o.5', was found to undergo a first-order reaction (t ~ = 125 min resulting in total disappearance of amino groups; aminolysis appeared to occur exclusively with the formation of the thiazine derivative (RE o.58 ). At pH 9.5, on tile other hand, the intramolecular rearrangement was accompanied by some hydrolysis, and chromatography permitted the detection of a small amount of the ninhydrin-positive hydrolysis product (RE o. 16) considered to be S-[(I-carboxy-2-N-ethylcarbamoyl)ethyll-L-cysteine: HO2C-CH (NH2) .CH2.S-CH(CO2H)-CH2CONHC~H 5. The addition product of cysteine with DDPM exhibited a half life period of 16 rain at pH 7.o. Chromatography of the reaction nfixture showed the presence of a single, ninhydrin-negative compound ; no hydrolytic product was observed. The thiazine derivatives formed in these re-arrangements have been previously isolated and characterized1, 2. The addition product of N-ethylmaleimide with homocysteine underwent a slow, first-order reaction at p H 9.5 (t i .... 168 rain). Chromatography of the reaction mixture showed a ninhydrin-positive hydrolysis product (RF o.3o) which was considered to be S-[(I-carboxy-2-N-ethylcarbamyl)ethyl]homocysteine. A very small amount of a ninhydrin-negative compound (RF o.7o), possibly a thiazine homologue, was also formed; in addition there was a trace of a second ninhydrin-positive product (RE o.16), probably formed by intermolecular aminolysis. Analogous results were obtained with the DDPM-homocysteine addition product at pH 7.0 (t, ....... 253 rain). The addition products of glutathione with N-ethylmaleimide and with DDPM, at pH values 9.5 and 7.o, respectively, underwent slow hydrolysis. Chronmtography of the reaction mixture of the glutathione-N-ethylmaleimide addition product did not reveal a ninhydrin-negative component or glutamic acid, which would have been released had intramolecular aminolysis occurred. A very small amount of a secondary product (RE o.I8), possibly formed by intermolecular aminolysis, was noted. The addition product of N-ethyhnaleimide with thioglycolic acid at pH 9.5 underwent slow hydrolysis (t I - 3Ol min). Addition of triglycine in equimolar concentration caused an increase in the rate of decomposition and chromatography of the reaction mixture revealed, in addition to the main product (/~F o.74) formed by hydrolysis, a small amount of ninhydrin-negative, sulfur containing compound (RF o.21), different from triglycine (RE o.Io). Elution of this product from the chromatogram, followed by hydrolysis at IOO° for 12 h in 6 M HC1, yielded only the free amino acid glycine. These observations are consistent with the occurrence of intermolecular aminolysis between triglycine and the imide ring of the maleimide- thioglycolic acid adduct. From the results recorded in Table I, it is seen that the infide ring of the Nethylmaleimide adducts exhibit striking differences in reactivity from tile imides derived from DDPM. This effect is no doubt related to the different inductive effects of the group attached to the imide nitrogen atom, the dinitrodimethylaminophenyl group being electron attracting and the ethyl group electron donating. Biochim. Biophys..4eta, i~,S (19(,S) 173

IXo

1 77

ACYLATION WITH CYCLIC IMIDES R.S CN--CO

R.S CH----CO

NO2

.CN3

CH2--CO

NO2

CH3

'N-.-CH2CH~ / CH2--CO

~N.(----~_N /\ / , •

N-ethylmoieimide odduct

DDPM adduct

In the series of reactions involving N-ethylmaleimide adducts, a comparison between the observed liberation of H + and the value calculated on the one hand for lOO% aminolysis or on the other for lOO% hydrolysis has enabled an assessment to be made of the relative contribution of each process to the overall reaction. The accuracy of the conclusions based on such an analysis depends on the degree of error involved in the measurement of the amino pK value and on the assumption that no change occurs in this value as a result of hydrolysis of the imide ring. The results obtained (Table III), interpreted within these limitations, confirm that intramolecular aminolysis is a reaction undergone readily only by the cysteine-N-ethylmaleimide adduct. From these experiments the following general conclusions can be drawn; intramolecular cross-linking of amino and thiol groups by maleimide reagents occurs only TABLE II[ COMPARISON OF AMINOLYTIC THIOL ADDITION COMPOUNDS

AND

HYDROLYTIC

REACTIONS

EXHIBITED

BY N-ETHYLMALEIMIDE--

R e a c t a n t s were incubated at p H 9.5; p K values were m e a s u r e d b y potentiometric titration.

Reactants

Thioglycolic a c i d - N - e t h y l m a l e i m i d e a d d u c t + triglycine ( I : I ) Cysteine-N-ethylma]eimide adduct Homocysteine-N-ethylmaleimide adduct Glutathione-N-ethylmaleimide adduct

p K of -NHs+ group

7.9 8.8 9.0 9.15

Theoretical number of H +formed from zoo molecules of reactant By hydrolysis

By aminolysis

Observed

IOO ioo IOO IOO

2.5 16.6 23. 7 30.7

97 15 88 95

when the groups are located in sterically favourable positions, and under optimal conditions the competing reactions of intermolecular aminolysis and hydrolysis are minor; DDPM adducts undergo both aminolysis and hydrolysis more readily than N-ethylmaleimide adducts; saturation of the olefinic double bond of N-ethylmaleimide or DDPM by addition of a thiol compound greatly increases the stability of the imide ring. EXPERIMENTAL

All melting points were uncorrected. Materials N-Ethylmaleimide (m.p. 45 °) was obtained from Sigma Chemical Corporation. Biochim. Biophys. Acta, 168 (1968) i73-I8O

178

D. (;. SMYTH, H. TUPPY

DDPM was prepared according to the method of W1TTER AND TuPPY '~. Cysteine hydrochloride naonohydrate was obtained frona Sigma Chemical Company, I>l.-homocysteine from Nutritional Biochenaicals Corporation, glutathione from Eastman Kodak Company, thioglycolic acid from Fisher Scientific Company, and glycylglycylglycine from Mann Research Laboratories Inc. N-Acetyl-L-cysteine was obtained as a generous gift from Dr. HANS T. CLARKE. Tile addition product of N-ethyhnaleimide with L-cysteine, 5-(1-ethyl-2,5-dioxopyrrolidin-3-yl)-L-cysteine, was prepared as described previouslyL2. The addition product of N-ethylnaaleinaide with thioglycolic acid was prepared according to the method of MARRIAN4. The addition product of DDPM with L-cysteine was prepared as described by WITTER AND TuPPY 2.

Chromatography The reaction products in samples of reaction mixtures were separated by ascending filter paper chromatography on Whatnaan No. I paper using butanol-acetic acid water (4:1:5, v/v/v, upper layer) as the solvent. The running time was about 12 h at room temperature. Chronaatogranas were dried in a current of air without the application of heat, then were sprayed with a o.I °/o (w/v) solution of ninhydrin in acetone or with an aqueous potassium iodide-chloroplatinic acid solution prepared as described by WINEGARD, TOENNIES AND BLOCK5, and were again allowed to dry at room temperature. Chromatography of reaction mixtures containing DDPM-thiol addition products was facilitated by the intense yellow colour exhibited by these conapounds.

S-(z-Ethyl-2,5-dioxopyrrolidin-3-3.,l)-DL-homocvstei~ze DL-Honaocysteine (135 nag, I nanaole) was dissolved in water (lO nal), initial pH 6.1, N-ethylnaaleinaide (125 nag, I nanaole) was added and tile flask was gently agitated to effect solution. After I h, acetone (20 nal) was added and a pale yellow solid (207 nag, na.p. 298-299 ° deconap.) which separated was filtered off and dried in a current of air. Recrystallisation of this material from an aqueous solution (4 nil) by cautious addition of acetone (20 nal) gave 196 nag of a white, crystalline product (na.p. 214 ° deconap.). A second crystallisation using the same conditions gave 171 nag of a product with the same melting point. Analysis: Calculated for C10Hs6N204S: C, 46.1; H, 6.2; N, IO.8(!/~>. Found: C, 46.0; 6.3; N, IO.8°/L.

S-( I-Ethyl-2 ,5-dioxopyrrolidin-3- yl )-glutathione Glutathione (307 nag, I mnaole) was dissolved in water (IO nal) and the solution was neutralised to pH 6.o by addition of IO nal of o.I M aq. NaOH. A solution of N-ethyhnaleinaide (125 nag, I nanaole) in water (IO nal) was added, and the solution was allowed to stand at room temperature for 3 h. The resulting solution was titrated with IO nal of o.i M HCI, then was evaporated in vacuo at 35 ° to about 5 mh Addition of acetone (2o nal) precipitated a white crystalline solid (438 nag, na.p. 205 ° decomp.) which was filtered off and dried. Recrystallisation from water (IO nal) bv addition of acetone (3° nal) gave tile pure product (383 nag, m.p. 206 ° decomp.). Analysis: Calculated for C16H24N4OsS" C, 44.4; H, 5.6; N, 12.9°..'i>. Found: C, 4 4 . 4 H, 5-7; N, I2.6%~.

Biochim. Bioph3,s..4cla, l(,S (19()¢S)

I 73

i,~o

ACYLATION WITH CYCLIC IMIDES

179

The addition product of DL-homocysteine with D D P M DL-Homocysteine (135 mg, I mmole) was dissolved in 2 ml of water and was mixed with a solution of DDPM (206 mg) dissolved in 3 ml of acetone. The mixture was left at room temperature for 15 min during which time some crystalline material separated. Crystallisation was made more complete b y addition of another 5 ml of acetone. The precipitate (154 mg) was filtered off and washed with acetone. From the filtrate, addition of ethyl ether and standing permitted the isolation of a further quantity (79 mg) of product. The combined products were suspended in water (4 ml) and were brought into solution by dropwise addition of I M HC1. To the acid solution a strong solution of sodium acetate was slowly added, drop by drop, with good stirring. The product separated out in finely crystalline form. Addition of sodium acetate was slowly continued until the supernatant fluid was pale yellow. The product was filtered off, and washed repeatedly with water (209 mg, m.p. indefinite). The purification procedure was repeated. I M HC1 acid was added to a suspension of the yellow material in water until it was almost completely dissolved. The undissolved material was filtered off. To the filtrate, sodium acetate solution was added with scratching until the p H was 4.5. The yellow crystalline precipitate was filtered off, washed m a n y times with water, followed b y acetone and ether. The product (155 rag) was dried at 780 for 2 h in vacuo. Analysis: Calculated for C16H19OsNsS: C, 43.5; H, 4.3; N, 15.9°/o . Found: C, 43.4; H, 4.6; N, I 5 . 9 ~o. The addition product of D D P M with N-acetyl-L-cysteine N-Acetyl-L-cysteine (163 mg, I mmole) was dissolved in 5 ml of acetone, and DDPM (206 mg, i mmole) dissolved in acetone (5 ml) was added. This solution was divided into two halves. One half was treated with ether, then with light petroleum and was allowed to stand for 12 h in a refrigerator. The material partially crystallised. When the precipitate was washed with ether containing acetone, some amorphous material (18 mg, m.p. 194-195 °) was left behind. The wash fluids and filtrate were combined and brought to dryness in vacuo at 35 °, dissolved in a small amount of acetone, and a small amount of ether was added until turbidity appeared; the solution was seeded and left to stand at room temperature. From time to time, more ether was added, and crystalline material (71 mg) was thus obtained. Recrystallisation was effected by dissolving in a small amount of hot acetone. After the solution had cooled, ether was slowly added in several portions at intervals of approximately 15 min. Most of the yellow colour was present in the crystalline product (63 mg) which after filtration, washing with ether, and drying at 780 for 2 h in vacuo had a m.p. of 192-193 ° decomb. Analysis: Calculated for C17H19NsOgS: C, 43.5; H, 4.1; N, 14.9%. Found: C, 43.7; H, 4.2; N, 15.1%. ACKNOWLEDGEMENTS The synthetic part of this work was carried out in the laboratories of Professor J. S. FRUTON, to whom the authors express their thanks, in the Department of Biochemistry, Yale School of Medicine, New Haven, Conn. Microanalyses were performed by Dr. S. M. NAGY,Department of Chemistry, Mass. Institute of Technology, to whom we wish to express our thanks.

Biochim. Biophys. Acta, 168 (1968) 173-18o

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D . G . SMYTH, H. TUPPY

REFERENCES 1 2 3 4 5

D. A. R. D. H.

G. SMYTH, A. NAGAMATSU AND J. S. FRUTON, J. Am. Chem. Soc., 82 (19oo) 4000. \VITTER AND H. TuPPY, Biochim., Biophys. Acta, 45 (196o) 429 . BENESCH AND R. E. BENESCH, Federation Proc., 19 (I96O) 78. H. MARRIAN, J. Chem. Soc., (1949) I515M. WINEGARD, G. TOENNIES AND R. J. BLOCK, Science, lo8 (1948) 5o(~.

Biochim. Biophys. Acta, 168 (1968) I 7 3 - t 8 o